Q J Med 1999; 92: 463-471
© 1999 Association of Physicians
CCR7 (EBI1) receptor down-regulation in asthma: differential gene expression in human CD4+ T lymphocytes
From the Molecular Medicine Unit, University of Leeds, St James's University Hospital, Leeds, 1 Genetics Directorate, GlaxoWellcome Medicines Research Centre, Stevenage, and 2 Respiratory Science, GlaxoWellcome R&D, Uxbridge, UK
Received 22 February 1999
Dr F. Syed, Molecular Medicine Unit, University of Leeds, Clinical Sciences Building, St James's University Hospital, Leeds LS9 7TF. e-mail: rmrfs{at}stjames.leeds.ac.uk
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Summary |
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Asthma is an inflammatory disorder, and the CD4+T lymphocyte plays a key role in mediating the inflammatory response. We used a high-density grid, hybridization-based, differential gene expression technology to analyse molecular mechanisms underlying in vivo CD4+ T-cell activation in both steroid-resistant asthma (SRA) and steroid-sensitive asthma (SSA). Hybridization of radioactively-labelled first-strand cDNAs prepared from different biological samples, to identical high-density gridded arrays of PCR amplicons derived from cDNA clone inserts immobilized on nylon membranes, was compared by phosphorimaging. Hybridization data were captured and processed using image analysis software that can identify the location and signal intensity of each hybridized cDNA. This produces a hierarchy of signals of differing intensities between the two grids, representing differential gene expression in the two different RNA samples. CCR7 (EBI1), a lymphocyte-specific G-protein-coupled receptor, was down-regulated in the CD4+ T cells of SRA and SSA non-atopic, compared to non-asthmatic non-atopic individuals. This observation is intriguing given that CCR7 and its ligand EBI1-Ligand Chemokine (ELC), may play a role in the migration and homing of normal lymphocytes. Also, TNFR2 is up-regulated in both SSA non-atopic and SRA atopic as compared to non-asthmatic controls. LAMR1 is down-regulated in CD4+ T cells of SRA compared to non-asthmatic individuals, irrespective of their atopic status. These could be general phenomena resulting from cytokine release.
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Introduction |
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Asthma is characterized by reversible airway obstruction, airway inflammation and bronchial hyper-responsiveness (BHR) to both non-specific irritants and specific triggers such as allergen.1 The airway inflammation leads to tissue destruction and airway wall remodelling involving epithelial disruption, smooth muscle and microvascular proliferation, basement membrane thickening and smooth muscle hypertrophy.2 Despite advances in the treatment of asthma, the overall mortality has not decreased significantly, while the prevalence and the severity of the disease are increasing.3 Airway inflammation is present in patients with mild, moderate or severe asthma.4 The mechanisms which underlie this bronchial mucosal inflammation are complex.
The CD4+ T lymphocyte plays a major role in the pathogenesis of asthma. It causes recruitment and activation of airway eosinophils and an increased IgE production from plasma cells.5,6 Corrigan et al.7 demonstrated that the percentages of CD4+ and CD8+ T lymphocytes, and the CD4/CD8 ratios in the peripheral blood, were similar in patients with acute severe asthma and control groups. In contrast, patients with acute severe asthma had significant increases, compared with controls, of three surface proteins associated with T lymphocyte activation: IL-2R, class II histocompatibility antigen (HLA-DR) and `very late activation' antigen, VLA-1.7 The IL-2R+ T lymphocytes were exclusively of the CD4+ phenotype.7 This provides evidence that activated CD4+ T lymphocytes can be identified in the peripheral blood of patients with acute severe asthma.7 The percentage of activated CD4+ T cells decreased with treatment and clinical improvement.7 CD8+ T cells in peripheral blood, from both asthmatics and controls, do not express IL-2R and VLA-1.8 Their expression of HLA-DR in asthmatics was also not increased.8 Robinson et al.9 showed that CD4+ T lymphocytes in bronchoalveolar lavage fluid and blood from asthmatic patients were activated, in comparison to controls. Within the asthmatic group, there was a significant association between CD4+ and CD25+ lymphocytes and asthma symptom scores. This clearly points to the CD4+ T cell being pivotal in the overall asthma inflammatory process, and shows the close correlation between CD4+ T-lymphocyte activation in peripheral blood and lung. Corticosteroids are the most effective anti-inflammatory drugs available to treat the majority of patients with asthma.10 However, not all asthmatics have a satisfactory clinical response to this therapeutic approach.11 Thus a better understanding of the factors underlying inflammation is required for new treatments to be devised.
We used a hybridization-based differential gene expression technology (developed within UK Genetics, GlaxoWellcome) to compare the molecular mechanisms underlying in vivo CD4+ T-cell activation in SRA and SSA. cDNA probes were prepared from total RNA isolated from CD4+ T cells of SRA, SSA and non-asthmatic individuals. These cDNAs were used separately to screen grids from a human spleen cDNA library (PL12), a human mixed tissue cDNA library with reduced redundancy (HS17) and an asthma custom cDNA grid containing known candidate genes. Hybridized filters were subjected to phosphorimage analysis. Image analysis, following normalization for any differences in cDNA probe activity, highlighted cDNAs which showed differential expression. These were sequenced, and homologies were identified in public databases.
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Methods |
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Patients
Three adult asthmatic patients, one SSA (non-atopic) and two SRA asthmatics (one atopic, one non-atopic) and six non-asthmatic controls (five non-atopic, one atopic), aged between 21 and 55 years, were studied. The number of patients studied in each category was different because of degradation of RNA from some patient samples. The non-asthmatic subjects were not taking any medication. All patients had a >15% increase in forced expiratory volume in 1 second (FEV1) following 200 µg inhaled salbutamol. In addition, the SSA patient had a >30% increase in FEV1 following a 4-week therapeutic trial of inhaled fluticasone propionate (FP), at 2000 µg/day. In contrast, the SRA patients had <15% improvement in FEV1 after this trial, but a >15% increase in FEV1 following 4 weeks of FP at doses ranging from 4000 µg to 6000 µg/day. Ethical approval for the study was given by the Leeds (East) Medical Ethics Committee.
Preparation of gridded cDNA libraries
PCR products amplified from 12 228 human spleen cDNA clone inserts (library from Gibco/BRL) were arrayed on to a positively-charged nylon membrane (Boehringer Mannheim) using a Genetix Q-bot. On separate membranes 12 228 PCR products were also arrayed from a second cDNA library HS17, which is a GlaxoWellcome proprietary cDNA library containing selected clones from human foetal skin, human foetal brain and a human B-cell library. An asthma custom grid, comprising cDNAs of genes known to be up-regulated in asthma, was prepared to use as a control (Table 1
).
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CD4+ T-cell isolation and RNA extraction
Peripheral blood mononuclear cells (PBMCs) were isolated from 50 ml of whole blood from each individual by density gradient centrifugation12 over Ficoll (Nycomed). CD4+ T cells were isolated13 from resuspended PBMCs through positive selection using CD4+ Dynabeads (M-450 CD4, Dynal), followed by depletion of CD8+ T cells using CD8+ Dynabeads (M-450 CD8, Dynal) as per the manufacturer's instructions.
To an aliquot of CD4+ T cells, we added 20 µl each of MsIgG1-FITC (Coulter), MsIgG1-RDI (Coulter) and MsIgGI-ECD (Coulter) antibodies. To another 50 µl cell aliquot, we added anti-CD3-FITC (a gift from Professor A.W. Boylston, University of Leeds), anti-CD4-RPE (Dako) and anti-CD8-ECD (Coulter). The cells were stained for 1 h at 4 °C in the dark and fixed with 500 µl fixing buffer [1xphosphate-buffered saline (PBS), 2% foetal calf serum (FCS), 2% normal human serum, 1% sodium azide, 1% formalin]. The purity of cells was determined by flow cytometry. Only cell preparations >98% homogenous for CD4+ were used in the study.
Total RNA was isolated from purified CD4+ T cells using the guanidinium thiocyanate-phenol-chloroform extraction method.14 Each sample was resuspended in 5 µl diethyl pyrocarbonate (DEPC)-treated distilled water (dH2O) (Promega) and 40 U RNAase inhibitor (Boehringer Mannheim) were added. Total RNA concentrations were determined by measuring optical density at 260 nm (Unicam UV/Vis spectrometer). Approximately 1 µg total RNA from each sample was checked for quality on a denaturing 1% agarose: 2.7 M formaldehyde:1x MOPS gel to detect potential RNA degradation.15
Probe labelling via reverse transcription
The five non-atopic, non-asthmatic RNA samples were pooled, resulting in four different overall categories. We labelled separately 5 µg total RNA from the RNA sample representing each category. The RNA was heated at 70 °C for 10 min with 1.44 µg (dT)15VN (Gibco/BRL). A mixture of 1xreaction buffer (Gibco/BRL), 10 mM DTT (Gibco/BRL), 0.5 mM dNTP mix (Promega), 40 U RNAase inhibitor, 4 µl [
-33P]dCTP (3000 Ci/mmol, Amersham), and 400 U of Superscript II (Gibco/BRL) was added. The reaction mixture was incubated at 42 °C for 90 min and the activity of the probe determined. First-strand cDNAs were purified using Sephadex G-50 spin columns (Pharmacia) as per the manufacturer's instructions. Quality of the complex probes was checked by polyacrylamide gel electrophoresis.
Hybridization of first-strand cDNA probes to gridded cDNA membranes
The cDNA array grid membranes for PL12, HS17 and the asthma custom grid (one each for each of the complex probes) were prehybridized in 10 ml Easy Hybridization (EasyHyb) solution (Boehringer Mannheim) at 45 °C for 30 min. Five µg poly(A)80 (Gibco/BRL), 10 µg human COT1 DNA (Gibco/BRL) and 435 µl EasyHyb were added to the purified probe prior to denaturation at 100 °C for 10 min. The probe solution was quenched at 45 °C for 90 min and hybridized to gridded cDNA filters for 3 days at 45 °C in a total volume of 10 ml EasyHyb. Three post-hybridization washes were performed (at 15 min intervals) in 50 ml 0.1xSSC, 0.1% sodium dodecyl sulphate (SDS) at 68 °C. Membranes were exposed to phosphorscreens for 2 days, and images captured using a Storm Scanner (Molecular Dynamics).
Processing and analysis of images
The captured images of these grids hybridized with the complex probes from asthmatic or non-asthmatic individual's CD4+ T cell RNA were processed using PC-based image analysis software. This software enabled us to mark the positions of the four corners of a hybridization image on the screen, identify the location of all the spots present, and predict where signals were not generated. This allowed each gridded cDNA coordinate to be located and accurately identified. The intensities of all spot coordinates were measured, and local background was subtracted. The intensity values of identically-located cDNA amplicon hybridization spots were compared to each other. The data was normalized statistically using the median spot intensity of each filter (excluding spots that were flooded or not found) for any differences in cDNA probe activity between filters. Flooded spots, poor replicates and spots of <1.2 fold difference were excluded from the comparison. This generated a tabulated list of differences, with the largest difference in cDNA hybridization at the top for each comparison. The top 20 cDNAs (those with the highest-fold difference) from each comparison list were visually checked to assess spot quality and reproducibility between the replicates in terms of intensity and ratio of difference before obtaining the plate and well locations of the respective amplicons showing the greatest intensity differences between hybridized asthmatic and non-asthmatic probes. Only cDNAs corresponding to the spots that fulfilled these criteria were picked for sequencing.
Sequencing
The 155 cDNAs which showed the greatest-fold difference were picked from the two non-custom libraries and grown overnight in LB medium. The cDNA inserts were amplified by PCR and products were visualized by agarose gel electrophoresis. These inserts were sequenced using fluorescently-labelled primers on an ABI 377 DNA sequencer. The resulting sequences were searched for homologies by screening the EMBL/Genbank and EST data bases using the BLAST search program.16
PCR of clones of interest
Vector primers flanking the inserted cDNA sequences (T7; dTAATACGACTCACTATAGGG and pCDM8R; dAGGCGCAGAACTGGTAG for CCR7 and LAMR1; CMV/T7; dGTCACACCACAGAAGTAAGGTTCC and CMV/SP6; dCGCCATCCACGCTGTTTTGACCTC for ITGB5) were used to amplify the clones of interest, and the products were visualized by agarose gel electrophoresis. Contaminating primers were removed by size separation of the PCR products using micron microconcentrators (Amicon) as per the manufacturer's instructions.
RNA dot blots
mRNA was isolated from the purified CD4+ T lymphocytes of SRA, SSA and non-asthmatic controls using the Message Maker kit (R&D Systems) as per the manufacturer's instructions.
For each of the three categories (SRA, SSA and non-asthmatic), a pooled mRNA sample was prepared (0.1 µg mRNA in total, with an equivalent contribution from five individuals per category). The pooled mRNA sample diluted in 1xSSC and formaldehyde was denatured at 60 °C for 10 min and then spotted onto a positively charged nylon membrane (Boehringer Mannheim) using a 96 well dot-blotting manifold (Gibco/BRL).
The quantity of mRNA loaded in each spot was assessed by overnight hybridization to end-labelled oligo(dT)15 (Gibco/BRL). To 20 pmol oligo (dT)15, 20 U polynucleotide kinase (NEB), 1xPNK buffer (NEB), 5 µl [
-33P]ATP (3000 Ci/mmol, Amersham), 37 µl dH2O were added and the reaction mixture was incubated at 37 °C for 30 min. Unincorporated label was removed using a Sephadex G-25 spin column (Pharmacia). RNA dot-blot membranes were prehybridized at 25 °C in DIG EasyHyb for 30 min. After discarding the prehybridization solution, 10 ml fresh EasyHyb was added to the hybridization bottle (Hybaid) and then 20 pmol probe. The hybridization was allowed to proceed overnight at 25 °C. The probe was discarded and phosphorimages were captured after washing four times (with 2xSSC at 25 °C for 5 min) and 4 h exposure to the phosphorscreen.
Template DNA (50 ng) (PCR amplicons of CCR7, LAMR1 or ITGB5 cDNA clones with contaminating primers removed) was radiolabelled ([-33P]dCTP, 3000 Ci/mmol, Amersham) using the Redi-prime kit (Amersham) as per the manufacturer's instructions. Unincorporated label was removed using Sephadex G-50 spin columns (Pharmacia).
The RNA dot blots were prehybridized at 45 °C for 30 min in 5 ml DIG EasyHyb. Having discarded this prehybridization solution, 5 ml fresh EasyHyb was added to the hybridization bottle (Hybaid). The probe was heated to 100 °C for 5 min and, after centrifugation, added to the dot-blot membrane to hybridize overnight at 45 °C. The probe was discarded and the membranes were washed three times for 20 min in 10 ml 0.1xSSC, 0.1% SDS at 68 °C. The dot blot was exposed overnight to a phosphorscreen prior to image capture.
ImageQuant software was used to calculate the two sets of hybridization signal densities with the local background subtracted, for each pooled mRNA sample dot-blotted. This was used to calculate the relative abundance of a given transcript per unit mRNA blotted.
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Results |
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Asthma custom grid
A custom grid comprising genes known to be aberrantly expressed in asthma was hybridized with each probe. This demonstrated class II major histocompatibility antigen HLA-DR up-regulation in SSA non-atopic, TNFR-2 up-regulation in SSA non-atopic and SRA atopic, C-JUN and C-FOS up-regulation in SSA non-atopic compared to non-asthmatic non-atopic individuals (Table 2
).
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cDNA library grids, PL12 and HS17
Replica grids from the PL12 and HS17 library clone amplicons were hybridized with cDNA probes prepared from SRA, SSA and non-asthmatic patients' CD4+ T lymphocytes. Proprietary PC-based image analysis software was used to compare the images for differences in spot intensity, generating an ordered list of the ratios of the differences between any two images compared.
Having tabulated the ratios of the differences in intensity, we obtained the plate and well locations of the top 20 cDNA clones (with the highest differences in the ratios of intensity) in each comparison list, to enable the relevant clones to be picked for sequencing. Of the 24 456 cDNA clones from both libraries, 155 clones were picked for sequencing (67 from the PL12 and 88 from the HS17 libraries, respectively). Of the two non-custom libraries, screened PL12 is more redundant than HS17. Of the 67 cDNA clones sequenced from PL12, 30 different cDNA species were identified. When 42 of the 88 clones from HS17 were sequenced, each represented a unique sequence.
Table 3 lists the differentially expressed genes identified of known function, which were carried through to dot-blot analysis, namely: Epstein-Barr-virus-induced gene (EBI1), also called chemokine receptor 7 (CCR7), down-regulated in SRA and SSA (non-atopic); the laminin receptor (LAMR1), down-regulated in SRA (atopic and non-atopic); and the integrin beta-5 subunit (ITGB5), down-regulated in SRA (atopic and non-atopic).
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Differential expression of CCR7
Comparison of the hybridization images from asthmatic and non-asthmatic samples showed differential expression of CCR7 in CD4+ T cells (Figure 1
). This was apparent in both the PL12 and HS17 gridded cDNA libraries, where CCR7 mRNA was down-regulated in both SSA and SRA (non-atopics) relative to non-asthmatic, non-atopic.
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RNA dot-blot analysis
RNA blot hybridization using pooled samples of mRNA from the CD4+ T cells of five individuals in each of the three clinical categories (SSA, SRA and non-asthmatic) were probed with a 1.3 kb PCR product from a cDNA clone for CCR7 and a 400 bp PCR product from a cDNA clone for LAMR1. This confirmed the differential expression of CCR7 and LAMR1 between asthmatic and non-asthmatic subjects (Figure 2
). Table 4 lists the abundance of each transcript per unit mRNA blotted. No signal for ITGB5 could be detected.
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Discussion |
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TopSummaryIntroductionMethodsResultsDiscussionReferences |
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High-density gridded array hybridization is potentially a powerful method of detecting differential gene expression between clinical samples. In this study, we have applied the approach to the detection of genes associated with CD4+ T cell activation (in vivo), in both SRA and SSA. A reduction in the abundance of mRNA coding for CCR7 was detected, which was confirmed by RNA dot-blot analysis. CCR7 was down-regulated in both the PL12 and HS17 cDNA libraries in both SSA and SRA.
Of the 11 asthma genes on the custom grid, only four showed differential expression between asthmatics and non-asthmatics (Table 2
). This could be due to the sensitivity of the detection technology, or possibly because their differential expression was below the 1.2-fold threshold.
CCR7 was originally identified as an Epstein-Barr virus (EBV)-induced gene.17 The encoded polypeptide is predicted to have eight hydrophobic domains likely to mediate membrane insertion. The first hydrophobic domain is believed to be a signal peptide, and the remaining seven transmembrane domains are characteristic of the G-protein-coupled receptor family. CCR7 is highly homologous to human high- and low-affinity interleukin 8 (IL-8) receptors at both nucleotide and amino acid sequence levels. Overall amino-acid identity among the three proteins exceeds 30%, excluding the putative CCR7 signal peptide sequence. However, the identity increases to 40% when comparing CCR7 to either of the two IL-8 receptors individually.17 This is the first G-protein-coupled receptor18,19 expressed exclusively in lymphocytes.17 Apart from its role in EBV infection, CCR7 is thought to be a tissue-specific mediator of cytokine effects. Its expression is strongly upregulated in B cells infected with EBV,17 and in CD4+ T cells infected with human herpes virus (HHV) 6 and 7.20 The extent of its homology to G-protein-coupled peptide receptors makes it probable that CCR7 is a receptor which transduces ligand-binding signals through heterotrimeric GTP-binding proteins (G proteins). The predicted action of CCR7 is mediated by G-protein activation of intracellular second-messenger pathways, involving the activation of effector molecules such as adenylate cyclase, cyclic AMP, phosphodiesterase, phospholipase C, or various ion channels.21
The mouse and human homologues of CCR7 have been cloned and the human gene mapped to chromosome 17q12–q21.2.22 This region of the genome has not been previously linked genetically to asthma, but is adjacent to the gene cluster for the CC chemokine family, at 17q11-q21.23 Chemokines are a group of approximately 70–90 amino-acid, structurally-related polypeptides that play important roles in inflammatory and immunological responses, primarily by virtue of their ability to recruit selected leukocyte subsets.24 Some chemokines also play roles in normal lymphocyte recirculation and homing.25,26 The chemokines are grouped into the CXC and CC subfamilies, on the basis of the arrangement of their two N-terminal cysteine residues. One amino acid separates the two cysteine residues in the CXC chemokines, whereas they are adjacent in the CC chemokines. Most CXC chemokines are potent attractants for neutrophils, whereas many CC chemokines are able to recruit monocytes and also lymphocytes, basophils, and/or eosinophils, with variable selectivity.24,27
The gene encoding the ligand for CCR7 has also been cloned20 and designated EBI1-Ligand Chemokine (ELC). This ligand is a novel human CC chemokine and highly specific for CCR7. It shows homologies to other CC chemokines, with 20–30% identity at the amino acid level.28 ELC is expressed most strongly in the thymus and lymph nodes, at an intermediate level in trachea, and at low levels in spleen.28 It maps to chromosome 9p13 rather than chromosome 17q11.2 where the genes for other CC chemokines are clustered.28 Chemokines and their receptors play a key role in leukocyte trafficking and extravasation of white blood cells into sites of inflammation.29,30 Hence ELC and CCR7 may play a role not only in inflammatory and immunological responses but also in normal lymphocyte recirculation and homing.21
The finding that EBV induces CCR7 expression is of interest in the light of recent findings that the genomes of herpesvirus saimiri,31 human cytomegalovirus,32 HHV633 and HHV734 all encode G-protein-coupled receptors homologous to chemokine receptors. A murine cytomegalovirus defective in open reading frame M33 (encoding a putative chemokine receptor) exhibited severely restricted growth in the salivary glands of infected mice.35 A chemokine receptor encoded by HHV8 was found to be constitutively active and to stimulate proliferation of transfected cells, making it a candidate viral oncogene.36 This suggests that virally-encoded, putative chemokine receptors play important roles in the infection and life cycle of herpesviruses, especially in vivo. ELC and CCR7 in EBV-infected B cells and HHV6- or HHV7-infected T cells may promote growth, protect from apoptosis and/or help migration into specific anatomical locations in vivo. Thus CCR7 synthesis is induced following EBV or other herpesvirus infection of B or T lymphocytes, respectively. Activation of CCR7 by its ligand leads to Ca2+ mobilization and T-cell chemotaxis. The reasons for down-regulation of CCR7 mRNA in both mild and severe asthmatic CD4+ T cells could reflect an excess of the ligand, resulting in down-regulation of the receptor as part of a negative feedback mechanism analogous to that for the ß2 adrenoceptor.37
More interestingly, there is now evidence for regulator T cells (Tr1), which are generated upon chronic activation of murine or human CD4+ T cells in the presence of IL-10.38 These Tr1 cells, which have low proliferative capacity, produce high levels of IL-10, low levels of IL-2 and no IL-4. This suggests that IL-10 induces the in vitro differentiation of a new regulatory CD4+ T cell subset which suppresses antigen-specific T-cell responses in vivo.38 Recent evidence suggests that there is selective expression of the eotaxin receptor CCR3 by human Th2-like cells.39 From this we might conclude that different subsets of Th cells express different chemokine receptors. Hence, Tr1 cells are likely to have their own specific chemoattractant signals, and anything that leads to a down-regulation of such signals may result in increased proliferation. CCR7 may be specific to Tr1, and down-regulation of its expression in asthmatics could account for the increased inflammation.
The studies described here were carried out on peripheral blood lymphocytes (PBL), and it might be argued that the reduced expression of CCR7 observed is because all cells with up-regulated expression have migrated to the lungs. This may mean that CCR7 is a proinflammatory receptor. Overall, our data points to a specific role for this novel CC ligand receptor in asthma. Whether this is related to cryptic viral infection or involves other factors which influence this specific homing pathway is not known. Validation of these finding at the protein level and in larger patient populations would justify further studies to fully elucidate the functions of this novel chemokine receptor.
The laminin receptor is a member of the integrin family of cell adhesion receptors.40 It is expressed on murine T lymphocytes.41 The alpha-6 beta-1 integrin (laminin receptor) is down-regulated by tumour necrosis factor alpha (TNF) and interleukin 1-beta in human endothelial cells. This is caused by the decreased expression of the alpha-6 subunit, whereas the synthesis of the beta-1 subunit remains constant.42 Hence the observation of down-regulation of the laminin receptor in asthma could be a general phenomenon due to the cytokines released by the activated cells in asthmatics.
In conclusion, the high-density array cDNA hybridization technology used in this study of asthma could be applied to any situation for monitoring changes in gene expression in disease states. The gene expression information uncovered in this way will help elucidate the molecular pathways underlying complex pathological conditions. These pathways may provide new therapeutic targets for the development of improved treatments of complex chronic disorders such as asthma.
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Acknowledgments |
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We thank the MRC for support for this project and Dr J.D. Issacs for his helpful comments on the manuscript.
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References |
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